Citation: Liao, Z.-H.; Huang, H.-T.;

Lin, Y.-R.; Chen, B.-Y.; Lee, Y.-F.; Lin,

Y.-H.; Chuang, C.-Y.; Nan, F.-H.

Natural Feed Supplements Improve

Growth, Non-Specific Immune

Responses and Resistance against

Vibrio alginolyticus in Lates calcarifer. J.

Mar. Sci. Eng. 2022, 10, 692. https://

doi.org/10.3390/jmse10050692

Academic Editor: Gabriella Caruso

Received: 30 April 2022

Accepted: 18 May 2022

Published: 19 May 2022

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Journal of

Marine Science and Engineering

Article

Natural Feed Supplements Improve Growth, Non-SpecificImmune Responses and Resistance against Vibrio alginolyticusin Lates calcariferZhen-Hao Liao, Huai-Ting Huang, Yu-Ru Lin, Bo-Ying Chen, Yi-Fan Lee, Yuan-Heng Lin, Chieh-Yuan Chuangand Fan-Hua Nan *

Department of Aquaculture, National Taiwan Ocean University, Keelung City 202301, Taiwan;smallhowhow@mail.ntou.edu.tw (Z.-H.L.); 29934001@mail.ntou.edu.tw (H.-T.H.);linyuru09@mail.ntou.edu.tw (Y.-R.L.); joey860812@gmail.com (B.-Y.C.); ian870414991106@gmail.com (Y.-F.L.);ak103t638@gmail.com (Y.-H.L.); 11033001@mail.ntou.edu.tw (C.-Y.C.)* Correspondence: fhnan@mail.ntou.edu.tw; Tel.: +886-2-24622192 (ext. 2910)

Abstract: This study aimed to investigate the effects of dietary natural feed supplement on the growthperformance, non-specific responses, and disease resistance in Lates calcarifer. Three commercialproducts (A, B, and C) containing a basal probiotic mixture were tested. Product A contained a basalmixture of Paenibacillus sp., Bacillus subtilis, Bacillus amyloliquefaciens, and Lactobacillus rhamnosus(107 cfu/g for each probiotic); product B contained additional Lactobacillus plantarum (1010 cfu/g);and product C contained additional soybean peptides (500 g/kg) and garlic powder (1 g/kg). Eachproduct was supplemented into subject diets at dosages of 1 or 2 g/kg (designated as the A1, A2,B1, B2, C1, and C2 groups, respectively). Following an eight-week trial, growth parameters (specificgrowth rate and feed conversion ratio), non-specific immune responses (O2

− production, phagocyticrate, and phagocytic index), and the results of a challenge test against Vibrio alginolyticus wereevaluated. The results show that all probiotic supplement groups exhibited an improvement ingrowth performance compared to the control group (non-probiotic diet). In terms of non-specificimmunity parameters, a significant improvement in O2

− production was found in the C2 group,whereas significant improvements in phagocytic activity were found in all the B and C groups. TheC2 group displayed optimal O2

− production, phagocytic rate, and phagocytic index results. For thechallenge test, the C groups showed higher Vibrio resistance than the other experimental groups andthe control group. These results suggest that product C, given at dosages of 2 g/kg, may serve as agrowth-promoting and immunostimulatory additive for the cultivation of Asian seabass.

Keywords: Lates calcarifer; probiotics; growth; non-specific immune response; Vibrio alginolyticus

1. Introduction

Disease outbreaks in fish and other economic aquatic species can cause huge lossesand hinder aquaculture development. For example, vibriosis, one of the most prevalentbacterial diseases, affects Epinephelus sp. groupers at every stage of development andcauses death in up to 50% of infected fish [1]. Traditionally, the control of vibriosis isachieved through antibiotics and disinfectants such as chloramphenicol and erythromycin,which are used against Vibrio harveyi infections in giant tiger prawns, and hypochloritesfor the treatment of vibrio-contaminated water [1]. However, such practices are usuallyaccompanied by a negative impact on aquatic animal health and environment [2]. Singleand mixed non-pathogenic probiotic products have been proven to be positive promoters ofaquatic fish growth, survival, and health. Previous studies have reported that incorporatingprobiotics into fish diets could improve intestinal microbiota, absorptive ability, digestiveenzyme activity, and expression levels of immune-related genes [3]. The administration of

J. Mar. Sci. Eng. 2022, 10, 692. https://doi.org/10.3390/jmse10050692 https://www.mdpi.com/journal/jmse

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probiotics is therefore considered an alternative method for disease control and preventionin various aquatic animal species [4].

Currently, various microorganisms such as Saccharomyces, Bacillus, Lactococcus,Micrococcus, Enterococcus, Lactobacillus, and Photorhodobacterium are considered to be pro-biotics in aquaculture [5]. Bacillus and lactic acid bacteria (LAB) are the most commonlyused probiotics for aquaculture functional feed formulations. B. subtilis produces a widerange of peptide antibiotics that are effective in inhibiting the growth of pathogens. Thisbacterium can also produce secondary metabolites, fine chemicals, and heterologous pro-teins for immune modulation [6]. LAB can produce molecules with bactericidal activityagainst intestinal pathogens or trigger immune responses in the host [7]. Different ad-ministration modes for probiotics have been proposed. Several studies have found thatthe co-administration of probiotics with prebiotics or plant products is beneficial. Garlic(Allium sativum) has many bioactive compounds. The various properties of garlic such asantibacterial, antioxidant and immunostimulant activity have made it suitable for combina-tion with probiotics in livestock farming [8,9].

Lates calcarifer, also known as barramundi or Asian seabass, is an important aqua-culture fish species commonly found in Australia and southern Taiwan [10]. In Taiwan,L. calcarifer are often cultivated under high-density conditions to increase profitability.However, this farming process and environment expose the fish to multiple stressors,increasing their susceptibility to disease. The present study aimed to develop a practicalframework for probiotic and plant product utilization in L. calcarifer. Using three novelcommercial probiotic products, respectively, containing a basal mixture of Paenibacillus sp.,Bacillus subtilis, Bacillus amyloliquefaciens, and Lactobacillus rhamnosus (107 cfu/g for eachprobiotic) (product A), additional Lactobacillus plantarum (1010 cfu/g) (product B), andadditional soybean peptides (500 g/kg) and garlic powder (1 g/kg) (product C), the effectsof dietary supplementation on the growth performance, non-specific immune responses,and disease resistance of Asian seabass were evaluated.

2. Materials and Methods2.1. Experimental Fish Rearing

Healthy Lates calcarifer were transported from a commercial fish farm to AquaticAnimal Center, National Taiwan Ocean University (NTOU). Fish from freshwater werethen acclimatized in seawater for two months prior to the experiment. Fish were keptin outdoor 8-ton fiberglass-reinforced plastic (FRP) tanks, and approximately 30% of thetank’s water is exchanged every day with fresh seawater. Fish received pelleted bass feed(Tairoun Products Company Ltd., Taipei, Taiwan; crude protein > 43%, crude lipid > 6%,ash < 16%, fiber < 3% and moisture < 11%) two times daily at amounts equivalent to 3% offish body weight.

2.2. Experimental Diets Preparation

A control diet (CT) obtained from Tairoun Products Co., Ltd. (Taipei, Taiwan) wascrushed for basal ingredients. Three commercial probiotic products (A, B, and C fromGreen Wonder Biotech Co., Ltd. (Taichung, Taiwan)) were, respectively, added into theCT feed at dosages of 1 or 2 g/kg of feed (referred to the A1, A2, B1, B2, C1, and C2groups). The feed powders were thoroughly mixed with α-starch (1 g/kg of feed) andreformed using a pelletizer. The diets were oven-dried at 40 ◦C for one day and then storedat 4 ◦C until use. Product A contained a basal mixture of Paenibacillus sp., Bacillus subtilis,Bacillus amyloliquefaciens, and Lactobacillus rhamnosus (107 cfu/g for each probiotic). Inaddition to the basal probiotic mixture, Lactobacillus plantarum (1010 cfu/g) was included inproduct B, and soybean peptides (500 g/kg) and garlic powder (1 g/kg) were included inproduct C.

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2.3. Feeding Trial

A total of 350 fish were randomly assigned to 7 groups placed in 14 outdoor 1-ton FRPtanks (25 fish/tank) with a seawater flow-through system. Fish were individually marked intail. After acclimation of 1 week, the feeding trail started and lasted 8 weeks. Fish (averageweight 50.79 ± 4.86 g) were fed with experimental diets twice daily at 09:00 and 16:00.Growth and death were observed and recorded every two weeks. Fish from each groupwere counted to calculate the survival rate (SR) and weighed individually to determine theweight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR). The growthparameters were calculated using the formula in our previous study [11]: WG = [(Finalweight (g) − Initial weight (g))/Initial weight (g)] × 100%; SGR = [ln (Final weight (g)) −ln (Initial weight (g))/Time duration (days)] × 100%; FCR = Feed intake (g)/(Final weight(g) − Initial weight (g)). During the experimental period, the water quality parameterswere monitored as follows: water temperature (25.5–30.5 ◦C), pH (6.9–8.1), dissolvedoxygen > 6.1 mg/L. NTOU’s Institutional Animal Care and Use Committee approved theexperiments (Approval number: 109012).

2.4. Immunity Tests

Three fish from each group were randomly sampled and dissected on ice. Isolation ofhead kidney leukocytes and following immune tests were performed using a previouslypublished method [12]. In brief, the head kidneys of the experimental fish were exsected,minced, and washed in Hanks balanced salt solution (HBSS; Gibco, Waltham, MA, USA).Cell lysates were passed through a 100 µm nylon mesh and the flow-through cell suspensionwas transferred to Percoll solution (Pharmacia, Stockholm, Sweden) with 30% and 50%density gradients. After being centrifuged at 400× g at 4 ◦C for 40 min, the leucocyteswere obtained at the interface. The leucocytes were then washed three times with HBSS,followed by adjustments of 5 × 106 cells/mL.

The measurement of O2− production was performed to evaluate the respiratory

burst activity. Moreover, 100 µL of leukocytes was loaded to a 96-well microplate. Acentrifugation of 800× g at 4 ◦C for 20 min was used to remove the non-adherent cellsand 100 µL of zymosan solution (0.1% in HBSS; InvivoGen, San Diego, CA, USA) wasadded to each well for 30 min at room temperature. After this step, the reaction mixturewas discarded before adding 100 µL of 0.3% nitro tetrazolium blue chloride solution (BioBasic Inc., Markham, Canada). After 30 min of incubation, the reaction was terminated byadding 100 µL of 100% methanol, followed by washing the cells with 70% methanol andair drying them at room temperature. In the last step, 120 µL of KOH (2 M) and 140 µL ofdimethyl sulfoxide (Gibco, Waltham, MA, USA) were added into each well and measuredat 630 nm with a microplate reader (BioTek, Santa Clara, CA, USA).

In the measurement of phagocytic activity, 100 µL of leukocytes was dispensed on aglass coverslip. After one hour of seeding, 100 µL of latex bead solution (3 × 106 beads,0.8 µm size; Sigma-Aldrich, St. Louis, MO, USA) in HBSS was added and incubated for onehour at room temperature. The cells were then washed three times with sterilized ddH2Oand fixed for 5 min using 300 µL of pure methanol. Finally, fixed cells were stained with5% Giemsa and air dried. Cells were observed under a light microscope (Olympus, Tokyo,Japan). The phagocytic rate (PR) was determined by phagocytic cell counts per 100 cells.The phagocytic index (PI) was calculated as the average counts of beads in phagocytic cells.

2.5. Challenge Test

V. alginolyticus was isolated from diseased fish and identified using 16S rDNA sequenc-ing. The bacterial stock was cultured overnight on tryptic soy agar (NuCel, Maisons-Alfort,France) with the supplement of 3% NaCl. Following that, the single colony was inoculatedto tryptic soy broth (NuCel, Maisons-Alfort, France) supplemented with 3% NaCl. Ashaker (100 rpm) was used to incubate the broth cultures for 4 h at 30 ◦C before centrifu-gation at 1000× g for 10 min at 4 ◦C. Eleven fish from each group were challenged withV. alginolyticus after the feeding trial. An additional 11 fish from the CT group were injected

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with sterile phosphate-buffered saline (PBS) as a negative control (CT-PBS group). A doseof 107 cfu/fish was injected into the abdomens of the fish using resuspended bacterialpellets dissolved in sterile PBS. Observations were conducted on the survival numbers ofeach group during a period of seven days.

2.6. Statistical Analyses

The experiments were conducted at least in triplicate, and data from growth andimmunity tests were presented as means ± standard deviations (SD). SAS software version9.4 was used for statistical analysis. Shapiro–Wilk tests and Levene test were performed toverify the normality of data and homogeneity of variance, respectively. A one-way ANOVAwith post hoc Tukey’s honest significant difference test was performed with significanceset at α = 0.05. Survival data for the fish challenge test were analyzed by Kaplan–Meiermethods using the SPSS software version 22.0. The Mantel–Cox test was used to evaluatethe differences between groups. Statistical significance (Sig.) is indicated by p values < 0.05.

3. Results3.1. Improvement of Growth by Probiotics and Plant Products

The growth performance after four weeks is shown in Table 1. Compared to the CTgroup, the WG, SGR, and FCR of all experimental groups were significantly improved(p < 0.05). Among the experimental groups, the A1 group showed the highest level ofgrowth after four weeks (SGR: 2.46 ± 0.07, FCR: 0.91 ± 0.03), but there were no significantdifferences between the experimental groups (p > 0.05). The growth performance after eightweeks is shown in Table 2. Compared to the CT group, the growth data of all experimentalgroups were improved. The WG and SGR of all experimental groups were significantlyimproved (p < 0.05), except in the B2 group. However, there were no significant differencesin FCR among the experimental groups (p > 0.05). Among the experimental groups, thehighest level of growth at eight weeks was achieved by the A2 group (SGR: 1.77 ± 0.10,FCR: 1.20 ± 0.10). No significant differences were found among the growth parameters ofthe experimental groups (p > 0.05). Moreover, the SR remained above 98% in each groupafter eight weeks of feeding, indicating that the three probiotic products are safe to use.

Table 1. Growth performance and survival of L. calcarifer fed with different natural feed supplementfor four weeks.

Groups Initial Weight(g)

Final Weight(g) WG (%) SGR (%) FCR FI SR (%)

CT 49.82 ± 4.88 a 84.42 ± 8.57 b 69.60 ± 1.91 b 1.89 ± 0.04 b 1.20 ± 0.03 a 41.55 100

A1 50.27 ± 4.89 a 100.04 ± 11.64 a 99.01 ± 3.93 a 2.46 ± 0.07 a 0.91 ± 0.03 b 45.49 100

A2 50.79 ± 4.59 a 96.26 ± 11.44 a 89.49 ± 3.08 a 2.28 ± 0.06 a 1.00 ± 0.04 b 45.54 100

B1 50.58 ± 5.00 a 98.63 ± 12.60 a 95.00 ± 1.71 a 2.39 ± 0.03 a 0.95 ± 0.02 b 45.56 100

B2 51.96 ± 4.26 a 98.31 ± 13.51 a 89.20 ± 2.04 a 2.28 ± 0.04 a 0.99 ± 0.03 b 45.82 100C1 50.92 ± 5.06 a 98.27 ± 11.16 a 92.98 ± 1.93 a 2.35 ± 0.04 a 0.98 ± 0.04 b 46.25 100C2 50.75 ± 5.34 a 97.25 ± 14.74 a 91.63 ± 4.01 a 2.32 ± 0.07 a 0.99 ± 0.03 b 45.95 100

F6,7 4.08 27.43 22.42 24.62 16.23p 0.0441 0.0002 0.0003 0.0002 0.0009

Note: Values are presented as mean ± SD (n = 50 for each group). One-way ANOVA and Tukey’s test wereused for analysis. Different superscripts indicate significant differences (p < 0.05) among means in the samecolumn. Abbreviations: WG: weight gain; SGR: specific growth rate; FCR: feed conversion ratio; FI: feed intake;SR: survival rate; CT: control; A1: A product 1 g/kg feed; A2: A product 2 g/kg feed; B1: B product 1 g/kg feed;B2: B product 2 g/kg feed; C1: C product 1 g/kg feed; C2: C product 2 g/kg feed.

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Table 2. Growth performance and survival of L. calcarifer fed with different natural feed supplementfor eight weeks.

Groups Initial Weight(g)

Final Weight(g) WG (%) SGR (%) FCR FI SR (%)

CT 49.82 ± 4.88 a 117.53 ± 14.21 b 136.59 ± 8.10 b 1.54 ± 0.06 b 1.32 ± 0.08 a 89.56 100

A1 50.27 ± 4.89 a 134.51 ± 18.28 a 167.57 ± 7.93 a 1.76 ± 0.05 a 1.23 ± 0.06 a 103.11 100

A2 50.79 ± 4.59 a 136.96 ± 19.06 a 169.66 ± 14.51 a 1.77 ± 0.10 a 1.20 ± 0.10 a 103.16 100

B1 50.58 ± 5.00 a 133.61 ± 19.24 a 164.23 ± 5.51 a 1.73 ± 0.04 a 1.24 ± 0.04 a 103.17 100

B2 51.96 ± 4.26 a 130.47 ± 19.32 ab 150.97 ± 8.53 ab 1.64 ± 0.06 ab 1.32 ± 0.08 a 103.43 100C1 50.92 ± 5.06 a 137.03 ± 17.48 a 169.10 ± 4.93 a 1.77 ± 0.03 a 1.21 ± 0.04 a 103.87 98C2 50.75 ± 5.34 a 134.30 ± 25.70 a 164.64 ± 20.99 a 1.74 ± 0.14 a 1.27 ± 0.16 a 105.13 100

F6,7 4.08 3.54 18.47 19.61 0.63p 0.0441 0.0021 <0.0001 <0.0001 0.7023

Note: Values are presented as mean ± SD (n = 50 for each group except C1 where n = 49). One-way ANOVA andTukey’s test were used for analysis. Different superscripts indicate significant differences (p < 0.05) among meansin the same column. Abbreviations: WG: weight gain; SGR: specific growth rate; FCR: feed conversion ratio; FI:feed intake; SR: survival rate; CT: control; A1: A product 1 g/kg feed; A2: A product 2 g/kg feed; B1: B product1 g/kg feed; B2: B product 2 g/kg feed; C1: C product 1 g/kg feed; C2: C product 2 g/kg feed.

3.2. Improvement of Immunity by Probiotics and Plant Products

Figure 1 shows the effects of probiotic supplements on the O2− production of L. calcarifer

leukocytes (F6,35 = 7.74, p < 0.001). The highest O2− production was found in the C2 group,

which showed a significant improvement compared to the CT group (p < 0.05). The B2 andC1 groups showed higher O2

− production levels than the CT group, but the differenceswere not significant (p > 0.05). No significant differences were found between the otherexperimental groups and the control group (p > 0.05). Figure 2 shows the effects of probioticsupplements on the PR of L. calcarifer leukocytes (F6,14 = 199.30, p < 0.001). Compared to theCT group, the PR of the B and C groups were significantly increased (p < 0.05), whereas thePR of the A groups were not (p > 0.05). The PR of the B1 group was significantly higher thanthat of the B2 group (p < 0.05). Moreover, the PR of the C groups were significantly higherthan those of other groups (p < 0.05). The C2 group had the highest PR, but showed nosignificant difference compared with the C1 group (p > 0.05). Figure 3 shows the effects ofprobiotic supplements on the PI of L. calcarifer leukocytes (F6,14 = 59.74, p < 0.001). Comparedto the CT group, the PI of the B and C groups were significantly increased (p < 0.05), but thePI of the A groups were not (p > 0.05). A higher PI was observed in the B1 group than in theB2 group (p < 0.05). In addition, the PI of the C groups were significantly higher than thoseof the B2 and A groups (p < 0.05). The PI of the C2 group was the highest, but showed nosignificant difference compared with the PI of the C1 group (p > 0.05).

3.3. Improvement of Disease Resistance by Probiotics and Plant Products

Disease resistance was evaluated using a V. alginolyticus injection challenge after thefeeding trial. The survival curve is shown in Figure 4A. The lack of mortality in the CT-PBSgroup at seven days after the challenge indicated that the fish could survive the injectionprocess. Surviving experimental groups C1, C2, and A1 showed respective survival ratesof 45%, 36%, and 18%, whereas no fish survived in the other challenge groups. Pairwisecomparison analysis of the survival between experimental groups is shown in Figure 4B.The survival probabilities of the C1, C2 and A1 groups were significantly higher than thatof the CT group (p = 0.001, p = 0.046, p = 0.027, respectively). There was no significantdifference between the three survival groups (p > 0.05).

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Figure 1. Superoxide anion (O2−) production of leukocytes taken from L. calcarifer fed with different commercial probiotics for 56 days. Values are shown as mean ± SD (n = 6). One-way ANOVA and Tukey’s test were used for analysis. Treatment groups with significant differences (p < 0.05) are indicated above the bars by different letters. Each commercial product was supplemented into control diets (CT) at dosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

Figure 2. Phagocytic rate (PR) of leukocytes taken from L. calcarifer fed with different commercial probiotics for 56 days. Values are shown as mean ± SD (n = 3). One-way ANOVA and Tukey’s test were used for analysis. Treatment groups with significant differences (p < 0.05) are indicated above the bars by different letters. Each commercial product was supplemented into control diets (CT) at dosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

Figure 1. Superoxide anion (O2−) production of leukocytes taken from L. calcarifer fed with different

commercial probiotics for 56 days. Values are shown as mean ± SD (n = 6). One-way ANOVA andTukey’s test were used for analysis. Treatment groups with significant differences (p < 0.05) areindicated above the bars by different letters. Each commercial product was supplemented into controldiets (CT) at dosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

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Figure 1. Superoxide anion (O2−) production of leukocytes taken from L. calcarifer fed with different commercial probiotics for 56 days. Values are shown as mean ± SD (n = 6). One-way ANOVA and Tukey’s test were used for analysis. Treatment groups with significant differences (p < 0.05) are indicated above the bars by different letters. Each commercial product was supplemented into control diets (CT) at dosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

Figure 2. Phagocytic rate (PR) of leukocytes taken from L. calcarifer fed with different commercial probiotics for 56 days. Values are shown as mean ± SD (n = 3). One-way ANOVA and Tukey’s test were used for analysis. Treatment groups with significant differences (p < 0.05) are indicated above the bars by different letters. Each commercial product was supplemented into control diets (CT) at dosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

Figure 2. Phagocytic rate (PR) of leukocytes taken from L. calcarifer fed with different commercialprobiotics for 56 days. Values are shown as mean ± SD (n = 3). One-way ANOVA and Tukey’s testwere used for analysis. Treatment groups with significant differences (p < 0.05) are indicated abovethe bars by different letters. Each commercial product was supplemented into control diets (CT) atdosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

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Figure 3. Phagocytic index (PI) of leukocytes taken from L. calcarifer fed with different commercial probiotics for 56 days. Values are shown as mean ± SD (n = 3). One-way ANOVA and Tukey’s test were used for analysis. Treatment groups with significant differences (p < 0.05) are indicated above the bars by different letters. Each commercial product was supplemented into control diets (CT) at dosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

3.3. Improvement of Disease Resistance by Probiotics and Plant Products Disease resistance was evaluated using a V. alginolyticus injection challenge after the

feeding trial. The survival curve is shown in Figure 4A. The lack of mortality in the CT-PBS group at seven days after the challenge indicated that the fish could survive the injection process. Surviving experimental groups C1, C2, and A1 showed respective survival rates of 45%, 36%, and 18%, whereas no fish survived in the other challenge groups. Pairwise comparison analysis of the survival between experimental groups is shown in Figure 4B. The survival probabilities of the C1, C2 and A1 groups were significantly higher than that of the CT group (p = 0.001, p = 0.046, p = 0.027, respectively). There was no significant difference between the three survival groups (p > 0.05).

(A)

(B)

Figure 3. Phagocytic index (PI) of leukocytes taken from L. calcarifer fed with different commercialprobiotics for 56 days. Values are shown as mean ± SD (n = 3). One-way ANOVA and Tukey’s testwere used for analysis. Treatment groups with significant differences (p < 0.05) are indicated abovethe bars by different letters. Each commercial product was supplemented into control diets (CT) atdosages of 1 or 2 g/kg and designated as the A1, A2, B1, B2, C1, and C2 groups.

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(A)

(B)

Figure 4. (A) Kaplan–Meier survivorship curves of L. calcarifer fed with different commercial probiotics for 56 days followed by V. alginolyticus challenge (n = 11 per group). Fish from the CT group were injected with sterile PBS as a negative control (CT-PBS group); (B) The pairwise comparisons between the groups were analyzed using the Mantel–Cox test.

4. Discussion Possible role of probiotics as beneficial mediators in aquaculture include the

improvement of nutrient utilization, microvilli surface area, gut microbial community, anti-stress enzymes activity, immune response, as well as the stimulation of decolonization of pathogen in host organisms [13]. The benefits of probiotic feed supplements on fish growth, specifically the components of the commercial products used in the present study, Paenibacillus sp. [14], B. subtilis [15], B. amyloliquefaciens [16], L. rhamnosus [17,18], and L. plantarum [19,20] were documented in previous literature. B. subtilis in particular has been proven to improve the growth performance of Asian seabass [21,22]. The dietary addition of B. subtilis (109 cfu/kg) has been shown to improve growth parameters, survival rates, body composition (increases proteins and decreases lipids), digestive enzyme activities (protease, amylase, and lipase) in the digestive tract, and hematological parameters (red blood cells, white blood cells, and hemoglobin levels). Moreover, soybean peptides, which were used in product C, were proven to benefit biological absorption and non-nutrition-related biological activities, displaying immunity, antibacterial, and antioxidant effects [23–25]. As for garlic powder, the other ingredient in product C, previous studies have shown that the inclusion of garlic (40 g/kg) in one’s diet can significantly improve the growth and feed utilization of Asian seabass [26]. Therefore, improvements in the growth performance of L. calcarifer can be achieved with the dietary supplements of these functional and safe materials. Further investigations

Figure 4. (A) Kaplan–Meier survivorship curves of L. calcarifer fed with different commercial probi-otics for 56 days followed by V. alginolyticus challenge (n = 11 per group). Fish from the CT groupwere injected with sterile PBS as a negative control (CT-PBS group); (B) The pairwise comparisonsbetween the groups were analyzed using the Mantel–Cox test.

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4. Discussion

Possible role of probiotics as beneficial mediators in aquaculture include the improve-ment of nutrient utilization, microvilli surface area, gut microbial community, anti-stress en-zymes activity, immune response, as well as the stimulation of decolonization of pathogen inhost organisms [13]. The benefits of probiotic feed supplements on fish growth, specificallythe components of the commercial products used in the present study, Paenibacillus sp. [14],B. subtilis [15], B. amyloliquefaciens [16], L. rhamnosus [17,18], and L. plantarum [19,20] weredocumented in previous literature. B. subtilis in particular has been proven to improve thegrowth performance of Asian seabass [21,22]. The dietary addition of B. subtilis (109 cfu/kg)has been shown to improve growth parameters, survival rates, body composition (increasesproteins and decreases lipids), digestive enzyme activities (protease, amylase, and lipase)in the digestive tract, and hematological parameters (red blood cells, white blood cells,and hemoglobin levels). Moreover, soybean peptides, which were used in product C,were proven to benefit biological absorption and non-nutrition-related biological activities,displaying immunity, antibacterial, and antioxidant effects [23–25]. As for garlic powder,the other ingredient in product C, previous studies have shown that the inclusion of garlic(40 g/kg) in one’s diet can significantly improve the growth and feed utilization of Asianseabass [26]. Therefore, improvements in the growth performance of L. calcarifer can beachieved with the dietary supplements of these functional and safe materials. Furtherinvestigations into the modulation of gut morphology, metabolism, and microbiota shouldbe conducted to address the growth augmentation.

The present data show that non-specific immune responses of Asian seabass wereimproved in the B and C groups, but not in the A groups, indicating that the componentsof product B can enhance phagocytic activity, and the ingredients of product C can enhanceboth phagocytic and respiratory burst activities. Previous literature has shown that thedietary addition of L. plantarum (106–1010 cfu/kg) for two weeks can increase phagocyticactivity in groupers (Epinephelus coioides) [20]. As no improvements in immunity werefound in the A groups, we theorize that L. plantarum (1 and 2 × 107 cfu/kg), an ingredientof product B, may contribute to the improvement of phagocytic activity in Asian seabass.Garlic (Allium sativum) has been widely used as an aquaculture feed additive [26,27] andcan be used as an immunostimulant to enhance resistance against bacteria, viruses, andparasites. Phagocytic activity and respiratory burst activity were improved in rainbowtrout (Oncorhynchus mykiss) fed garlic powder (5 g/kg) for two weeks [28,29]. In addition,the dietary administration of raw garlic or garlic powder (10–80 g/kg) for eight days hasbeen shown to enhance phagocytic and respiratory burst activity in E. coioides [30]. Thepresent data show that trace amounts of garlic powder contained in product C may play animportant role in improving the phagocytic and respiratory burst activity of Asian seabass.

The challenge test showed that the C groups had the best disease resistance againstV. alginolyticus, which cause heavy mortalities in Asian seabass farming [31]. The C groupsalso had the highest levels of immunity improvement. A previous report showed that rain-bow trout (Oncorhynchus mykiss) fed garlic powder (5 g/kg) for 14 days displayed improvedresistance against Aeromonas hydrophila [29]. Increase in resistance against Vibrio harveyiwas also found in L. calcarifer fed garlic powder (5 g/kg) for 14 days [32]. The antibacterialproperties of garlic prevent the colonization of pathogenic bacteria in the intestine andinfluence immune responses by providing an ideal environment for the proliferation ofbeneficial probiotics [27]. It was noted that garlic extract can serve as prebiotics for LAB [33].A 4% garlic extract is the optimal concentration that can effectively promote growth ofLactobacillus acidophilus. Moreover, dietary supplementation with soybean peptides replac-ing 50% of fish meal was shown to reduce mortality in yellow catfish (Pelteobagrus fulvidraco)after A. hydrophila infection [34]. Soybean peptides may improve immune responses andenhance the disease resistance of yellow catfish. This improvement of immune responsewas also reported in Japanese flounders, suggesting that the dietary supplement of 10%soybean peptide could enhance the survival of Paralichthys olivaceus under heat stress [35].Product C contains 50% soybean peptides and 0.1% garlic powder. We therefore speculate

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that the dietary additions of soybean peptides (0.5 and 1 g/kg) and garlic powder (0.001and 0.002 g/kg) can effectively improve Vibrio resistance of Asian seabass.

Research on the use of probiotic mixtures and their effects on fish growth and immu-nity is presently limited. The immune efficiency and disease resistance of probiotics areassociated with the strain, viability, dosage, and the duration of administration [36]. It hasbeen reported that the dietary administration of a mixture of B. subtilis and L. acidophilusprovided better disease protection for Oreochromis niloticus Nile tilapia than the use of asingle probiotic [37]. However, the antagonistic interactions among probiotics should becarefully evaluated prior to the administration of probiotic mixtures for different aquaticanimals. It has been reported that L. plantarum can inhibit the growth of B. subtilis anddecrease probiotic efficiencies in Scylla paramamosain mud crabs [38], which could explainwhy the survival of the B groups was worse than that of the A groups in the presentchallenge test. In other words, L. plantarum may alleviate the beneficial effects provided byB. subtilis.

In sum, the present work indicates that the dietary administration of combined pro-biotics and plant products have positive effects on the growth performance, non-specificimmune responses, and the pathogen resistance of the Asian seabass. The appropriatecombination of multispecies probiotics and other bioactive substances may have comple-mentary effects on fish aquaculture. This study provides a successful example of the use ofnatural feed supplements in sea bass mariculture.

Author Contributions: Conceptualization, Z.-H.L. and F.-H.N.; methodology, H.-T.H.; formal analy-sis, B.-Y.C.; investigation, Y.-F.L., Y.-H.L. and C.-Y.C.; writing—original draft preparation, Z.-H.L.;writing—review and editing, Y.-R.L.; supervision, F.-H.N.; project administration, Z.-H.L.; fundingacquisition, F.-H.N. All authors have read and agreed to the published version of the manuscript.

Funding: This research was supported by an industry–academia cooperation project, grant number(NTOU 109B2_1201).

Institutional Review Board Statement: The animal experiments were approbated by the NTOU’sInstitutional Animal Care and Use Committee (approval number: 109012).

Informed Consent Statement: Not applicable.

Data Availability Statement: The corresponding author of this study can provide the present dataupon request.

Acknowledgments: We thank Green Wonder Biotech Co., Ltd., for providing the probiotic productsand Po-Tsang Lee for providing the pathogen strain used in the challenge study.

Conflicts of Interest: The authors declare no conflict of interest.

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